The present invention relates to the electrophoresis separation and photodetection apparatus for the DNA or RNA, especially to the fluorescence detection type electrophoresis apparatus suited to detect the fluorescence having different wavelengths emitted from the samples which are labeled with a plurality of fluorophores.
The gene diagnosis and the analysis of human genome require a quick analysis of many samples. The conventional method used for DNA detection including the base sequence determination for DNA has been autoradiography, whereby the DNA is labeled with the radioactive element, and the pattern of the DNA fragment subjected to electrophoresis gel separation according to its length is photographed and read out. Furthermore, automatic DNA sequencers have recently been developed, whereby the DNA fragment is labeled with the fluorophore to provide real time detection (Nature, Vol. 321, 1986, pp. 674-(679)).
These sequencers use a plurality of fluorophores having different emission wavelengths to distinguish the four types of bases from one another or classifies the types of DNA to be detected. Namely, the means to identify signals emitted from a plurality of fluorophores have been proposed, including;
(1) a system which uses the rotary filter provided with four types of filters which selectively transmit the light emitted from the fluorophores installed on the front of the detector (Nature, Vol. 321, 1986, pp. 674-679),
(2) a system which uses the image splitting prism (Japanese Patent Application Laid-open 2-269936 and U.S. Pat. No. 5,062,942), and
(3) a system which uses the wavelength dispersion prism (Japanese Patent Application Laid-open 1-116441 and U.S. Pat. No. 4,832,815).
However, the multicolor fluorescence detection system using the rotary filter has a problem; the increased number of fluorophores leads to reduced time to measure each fluorophore, hence reduced sensitivity. The conventional method of using the latter image splitting prism permits identification of about four types of fluorophores excited by one laser. However, effective excitation by one laser is possible up to two types of fluorophores generally; use of more than two types of fluorophores requires two or more excitation light sources in practice. As a result, use of two or more excitation light sources have often caused overlapped bands between the second excitation light wavelength and the fluorescent wavelength excited by the first excitation light. Especially, greater numbers of fluorophores tend to increase the frequency of overlaps between the excitation light wavelength and emission wavelength of the fluorophore disadvantageously in measuring. Namely, one laser is used generally because of the measuring problem. However, in this case, the multicolor fluorescence labeling method which detects the fluorescences with different wavelengths from samples labeled with these plurality of fluorophores has the problem of poor excitation efficiency with respect to some of the fluorophores, and of poor sensitivity in general; this is because of lack of the laser system which ensures effective excitation of all the fluorophores. At present, the apparatus using said method is placed on the market by Applied Biosystems Inc. in the U.S.; it has 24 migration lanes. This apparatus permits one measurement in ten to twelve hours, and allows only twenty-four samples at one time.
Another known method uses the procedure of labeling the DNA by a kind of fluorophores, thereby identifying the terminal species according to the difference of the migration lanes. However, this single-color fluorescence labeling system using one fluorophore label permits one measurement in five to six hours, allowing eight to ten samples at one time; the number of samples measured per day (throughput) is limited to only 16 to 20, when two daily measurements are assumed.
As discussed, the conventional DNA sequencers have been far from satisfactory in ensuring a quick, simultaneous analysis of many samples.